Study of Phytochemical Attributes of Ashwagandha Ghrita from Desi Cow Milk

In rural areas worldwide, more than 80% of people use plant-based traditional medicines such as Ayurveda, Unani and traditional Chinese medicine (Majeed, 2017). Ashwagandha (Withania somnifera (L.) Dunal) is an ancient medicinal herb used in Ayurvedic and Unani medicine. With over a hundred years of experience in cultivating, extracting and distributing ashwagandha, the world’s largest producer of this herb is India. Other countries are consuming Ashwagandha in the form of pills, capsules and other formulations from India. (Khabiya et al., 2023). There are various pharmacological uses for this herb, including analgesics, antioxidants, anti-inflammatory, antidepressants, antimicrobials, anticonvulsants, anxiolytics and hypnotics, cardioprotective, nootropics, etc. (Yadav et al., 2024; Langade et al., 2023).
       
Ashwagandha roots are used in Ayurvedic medicine for their therapeutic properties. Ashwagandha roots contain phenolics, steroids, saponins, alkaloids, glycosides and volatile oils in addition to several hundred traditional phytochemicals (Yadav and Rai, 2018a; Naveed et al., 2022). Besides having antioxidant and anti-inflammatory properties, these compounds also play a crucial role in producing therapeutic effects (Mandlik et al., 2021). As a result, the usage of medicinal plants is becoming more widespread than it was previously, ushering in a new era in the treatment of several chronic illnesses, such as obesity, diabetes and hypertension (Choudhary et al., 2023).
       
To maintain and improve health, nutrition and immunity, health-conscious consumers have shown a growing interest in making use of the therapeutic and functional properties of herbs. Spices and herbs were traditionally considered to be medicinal foods (Rani et al., 2023). The trend of fortifying dairy products with these herbs to take advantage of their therapeutic and functional properties has also gained attraction (Paswan et al., 2021; Yadav and Rai, 2018b). Milk and dairy products are increasingly popular due to their reputation as a nearly complete source of nutrition, often described as a “nutrient reservoir”(Guptaet_al2022). Ghee is one of the crucial components of the Ayurvedic medical system. Ghee is the Indian term for clarified butter fat, which is made by boiling it off. Buffalo and cow milk, either individually or in combination, are typically used to make it. In India, approximately 50-55% of the country’s total milk production is utilized in the creation of traditional dairy products (Singh et al., 2023). Ghee is made from about 30 to 35 per cent of the milk produced in India (Laghari et al., 2023). Ghee and butter, in particular, can capture all of the therapeutic and functional properties of the integrated bio-actives without altering their qualities (Pandhi et al., 2021).
       
In the current millennium, the dairy industry is shifting from a focus on bulk commodities to an emphasis on innovative and flavorful value-added products. This transition is marked by the development of niche segments that prioritize unique taste, color and visual appeal (Sahana and Vijayalaxmi, 2024). Ghee serves as a basis for the Ayurvedic medication ghrita, which dissolves, extracts, or retains the active medicinal ingredients. Ghee preparations with added medicinal properties, known as ghritas, are made with the fat-soluble parts of the components (Prajapati et al., 2017). Dairy products could provide consumers with not just the usual nutritional benefits but also various additional health benefits. The purpose of this study was to develop herbal ghee with cow’s milk and ashwagandha ghrita. The product was also assessed for characterization, antioxidant, phenolic activity and sensory characteristics.

The experimental research was conducted in the laboratory of the Department of Dairy Science and Food Technology at the Institute of Agricultural Sciences, (BHU). Cow milk was purchased from dairy farm at BHU Varanasi. Ashwagandha root was procured from the local market of Varanasi. All the chemicals used during the study were of analytical grade and were procured from Hi Media Laboratories, Pvt. Ltd., Mumbai, India.
 
Preparation of ashwagandha kwath
 
The process outlined in the Ayurvedic Sarsangraha was followed to make ashwagandha kwath (Gurav et al., 2020) as shown in Fig 1. Ashwagandha roots were dried in the sun for a day to remove moisture. They were then crushed into coarse powder using a mortar and pestle. The coarse powder was separated and the remaining ashwagandha roots were further ground into fine powder using a mixer and grinder. Approximately 200 g of the coarse ashwagandha root powder was measured and soaked overnight in four liters of water. The next day, the container with the soaked ashwagandha was heated on a gas burner until the liquid was reduced to half its original volume, which took about 6 hour. This liquid was then filtered and set aside.
 

 
Preparation of ashwagandha ghee
 
The methods for the formation of ghee and preparation of the sample were according to De (2005) with slight modifications are shown in Fig 1 and 2. In a separate pot, several liters of milk were boiled. To this boiling milk, 200 g of ghee and 50 g of finely powdered ashwagandha were added. In addition to the liquid, the one-liter decoction (liquid) prepared earlier was added too. The mixture was boiled on low heat for an additional 2 hour. After boiling, it was left to cool overnight. The next morning, heating was resumed and occasional stirring was done. During the heating, a frothy layer formed on the surface of the mixture and the milk started to curdle, forming a solid consistency after about 6 hours of continuous boiling. By the 10^th hour of reduction, a cohesive mud-like paste had formed at the bottom of the container. Continuous stirring was done to prevent the paste from charring. As time passed, around the 12^th hour of stirring, the frothy layer on the surface began to disappear. We kept on heating the mixture until every bit of water had evaporated from the ghee and the ghee started to separate from the paste. As it reached the final stages, the ghee became clear and transparent and there were no bubbles or froth left. To make sure that all the water had completely evaporated, we took a small amount of the paste and burned it. If there were no crackling sounds, it showed that all the water had been successfully removed. This step was crucial to confirm that the ghee was pure and didn’t contain any remaining water.
 

 
Characterization of ashwagandha root powder
 
Using standard AOAC procedures (2007), chemical analysis of dehydrated ashwagandha (Withania somnifera) was conducted to approximate the concentration of moisture, ash, fat, protein and fiber. The calorimetric method was used to determine iron, while the titration method was used to determine calcium. Using the 2, 6-Dichlorophenol dye method, Vitamin C was determined.
 
Characterization of ghee
 
Moisture and fat
 
The moisture and fat percent of Ashwagandha ghee were determined by the AOAC (2007) method.
 
Butyro refractometer, reichert-meissl and polenske value
 
The butyro refractometer (BR) was used to determine the butyro refractometer (BR) reading of the ghee samples. Using the conventional procedure by BIS (1981), the Reichert-Meissl (RM) and Polenske values of ghee samples were calculated.
 
Oxidative stability (Peroxide Value)
 
Ghee peroxide value was ascertained using Lea’s method, which is described in the BIS method (IS:3508, 1966), to assess oxidative stability.
 
Vitamins and cholesterol
 
The Vitamins and Cholesterol of ghee were determined by the AOAC method as described in (AOAC, 2000).
 
Sodium, potassium, calcium and iron
 
With slight modifications, the procedure outlined by (Vasanth et al., 1971) was used to ascertain the mineral content of ghee.
 
Quantification of antioxidant and phenolic properties of ghee
 
DPPH was calculated using the scavenging activity method developed by (Patel et al., 2022). 200 µl of prepared samples were analyzed at a wavelength of 517 nm for this study. A UV-VIS spectrophotometer (Shimadzu, Japan) was used to measure the absorbance.
   
Where,
A0 = Absorbance of the control.
A1 = Observed final absorbance of the extracted sample at the wavelength of 517 nm.
In the blank experiment, 95% Ethyl acetate was used
 
Total phenolic content
 
The total phenolic content (TPC) was determined according to the method described by (Krawitzky et al.,2014) with slight modifications. For this, 100 µl prepared sample was taken after that, a UV-VIS spectrophotometer (Shimadzu, Japan) was set to detect absorbance at 760 nm wavelength. Finally, the total phenolic content in the sample was expressed in micrograms of Gallic acid equivalent (GAE) per milliliter (ml) of the extract. This measurement helps assess the concentration of phenolic compounds, which are important antioxidants, in the sample.
 
Statistical analysis
 
To measure the phenolic, antioxidant and characterization qualities of ashwagandha ghee, the experiment was carried out in triplicate. To ascertain the significance of sample differences, OP Stat software was used. Significance levels were decided at (p<0.05).

Chemical composition
 
The comparative chemical composition of control ghee and ashwagandha ghee is presented in Table 1. The control ghee and ashwagandha ghee had almost the same moisture content, 0.20% in control ghee and 0.18% in ashwagandha ghee. The fat content was very high and was comparable in both ghees, with control ghee containing 99.85% and Ashwagandha ghee containing 99.82% fat. The free fatty acid content of control ghee was 0.20% compared to 0.40% of Ashwagandha ghee. The Butyro refractometer Reading of the control ghee was 41 while the Ashwagandha ghee was 42. The Reichert-Miesel value of the control ghee was 31, while the Ashwagandha ghee was slightly higher. Both ghees had similar Polanske-value, control ghee was 1.18 and Ashwagandha ghee had 1.19. The percentage of DPPH inhibition in control ghee (75.6%) was lower compared to Ashwagandha ghee (83.98%). The total phenolic content of ashwagandha ghee was 62.85 μg GAE/ml, while control ghee showed no detectable amount. The phytosterol content of ashwagandha ghee was 0.33 mg/g. The peroxide value of control ghee was 0.75, whereas ashwagandha ghee had a slightly higher value of 0.80.
 

       
A comparison between the fat content of regular cow ghee and ghee enriched with ashwagandha can reveal any changes in the lipid profile brought about by the herbal enrichment. General research on the nutritional profiles and composition of ghee was discussed by (Kumar et al., 2018) which proves that supplementation with herbs in ghee reduces the moisture content. Readings from a Butyro refractometer at 40°C are used to determine the composition and purity of ghee. It evaluates how much solid fat is in the ghee (Gandhi and Lal, 2017). Variations in this value between regular cow ghee and ghee enhanced with ashwagandha could be a sign of changes in the composition and crystalline structure (Ramya et al., 2019). One metric used to evaluate the volatile fatty acid concentration of ghee is the Reichert-Meissl value. It is a sign of the ghee’s quality and freshness (Nekera et al., 2023). Variations in the amounts of volatile fatty acids between the enriched and regular ghee can provide information on the stability and possible oxidation of the goods (Kumbhare et al., 2021). Studies on volatile fatty acids in dairy products can be cited to support information on the Reichert-Meissl value and its implications for the quality of ghee (Gandhi et al., 2014). The Polenske value evaluates the amount of volatile fatty acids while also taking the existence of unsaturated fatty acids into account, much like the Reichert-Meissl value. Variations in the fatty acid composition and oxidative stability between the two forms of ghee may be indicated by differences in Polenske values (Ahmed et al., 2020). As with the Reichert-Meissl value, pertinent research on fatty acid content and oxidative stability might be consulted when discussing the Polenske value (Ahmed et al., 2020). The peroxide value is a measure used to determine the extent of primary oxidation in fats and oils. Increased oxidative stress is indicated by elevated peroxide levels (Gotoh and Wada, 2006). Peroxide readings from regular cow ghee and ghee enhanced with ashwagandha can be compared to provide insight into the products’ oxidative stability. Research on lipid oxidation in dairy products can be cited to support a thorough discussion of peroxide value (Pawar et al., 2014).
       
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay quantifies a material’s antioxidant capacity (Shahidi and Zhong, 2015). When comparing ashwagandha-enriched ghee to regular cow ghee, a higher percentage of DPPH inhibition would suggest that the addition of ashwagandha has boosted the antioxidant ability due to the presence of Withaferin and other compounds. Research studies on the antioxidant qualities of herbs and functional foods were cited to support discussions on the DPPH assay and antioxidant activity (Gurav et al., 2020).
       
Phenolic chemicals enhance a product’s capacity to function as an antioxidant. A comparison of total phenolic content can reveal information about the overall richness of antioxidants (Piluzza and Bullitta, 2011); greater values in ghee supplemented with ashwagandha suggest possible health benefits. Studies on the phenolic composition of herbs and their contributions to antioxidant capacity were used to support information on total phenolic content and its significance (Pawar et al., 2014).
 
Nutritional profile
 
The nutritional profile of control ghee and ashwagandha ghee is presented in Table 2. The micronutrients such as sodium, potassium, calcium and iron were found to be high in ashwagandha ghee as compared to control ghee, while the Vitamin A, cholesterol and energy value were found to be high in control ghee as compared to ashwagandha ghee.
 

       
When compared to the control ghee, ashwagandha ghee has noticeably higher levels of salt, potassium, calcium and iron. This could be explained by ashwagandha’s natural mineral composition, which is high in important minerals (Nekera et al., 2023). Ashwagandha ghee’s improved mineral composition may help it fulfill its potential as a nutrient-dense dietary item. When compared to ashwagandha ghee, the control ghee has a higher vitamin A content. The variance might result from the unique ways that ashwagandha is extracted and processed, which could affect how much of certain vitamins are retained (Singh et al., 2011). To clarify the impact of ashwagandha on the vitamin makeup of ghee, more investigation is required.
       
Ashwagandha ghee has less cholesterol than ghee which is used as a control. This result is consistent with earlier research that suggested some herbal extracts, such as ashwagandha, may have the ability to decrease cholesterol. Ashwagandha ghee’s possible cholesterol-lowering qualities may be a factor in its advantages for cardiometabolic health (Sharma et al., 2010). Ashwagandha ghee has a somewhat lower total energy content than control ghee. This could be explained by the unique makeup of ashwagandha and how it might affect the ghee product’s total energy density (Singh et al., 2011). To fully comprehend the impact of ashwagandha on energy metabolism, more research on the metabolic effects of ghee is necessary (Sharma et al., 2010).
       
According to comparative nutritional research, ashwagandha ghee has a unique nutritional profile that is enhanced with important minerals and may have certain health advantages. Variations in vitamin content, however, call for additional research. The benefits of Ashwagandha ghee as a functional food are evident in its impact on cardiovascular health and energy balance. These benefits are underscored by the notable differences in cholesterol levels and energy content.
 
Shelf-life evaluation by sensory and antioxidative parameters
 
The sensory parameters and several antioxidative parameters of control ghee and ashwagandha ghee during the storage period have been presented in Table 3. The flavor score of control ghee (9.01±0.02) was found higher than ashwagandha ghee (8.80±0.02). On the opposite, the texture score was slightly higher in ashwagandha ghee (24.55±0.10) as compared to control ghee (24.25±0.02). The score of color  and appearance was higher in control ghee (9.05±0.02) as compared to ashwagandha ghee (8.87±0.02). There were no significant differences in the score of overall acceptability. The score of overall acceptability was slightly higher in ashwagandha ghee (94.20±0.02) as compared to control ghee (94.15±0.02). From the study, it was revealed that as the days pass during storage the antioxidant values in the form of FFA, DPPH and peroxide values decrease significantly. The same pattern was also observed for the organoleptic parameters as well.
 

       
Sensory evaluation is a way to scientifically study and understand how our senses (like sight, smell, taste, touch and hearing) experience things, such as food or materials (Tzia et al., 2023). It helps us to measure, analyze and make sense of what we feel, see, smell, taste, or hear when we interact with something. The temperature at which fat (ghee) is stored significantly impacts both sensory acceptance and oxidative degradation. The special taste of ghee comes from a mix of different compounds created when it is made (Pena-Serna and Restrepo-Betancur, 2020). These compounds, like free fatty acids, carbonyls and lactones, give ghee its unique aroma (Bumbadiya et al., 2023). Ghee takes on a different flavor and aroma when produced with Ashwagandha containing certain bioactive chemical compounds. Our findings agreed with Rajnikant, (2005) findings that the flavor score of ghee significantly decreased with an increase in the amount of arjuna herb in cow ghee. The addition of ashwagandha may be the reason for the minor reduction in flavor intensity in Ashwagandha Ghee, as shown by the lower score when compared to control ghee (Gurav et al., 2020). The unique flavor profile of the herb may affect the ghee’s overall flavor. Research indicates that the perception and acceptance of flavor may be affected by herbal additions. It’s interesting to note that Ashwagandha Ghee has a little better texture score than Control Ghee. This outcome might be the result of Ashwagandha’s special makeup, which could alter the ghee’s texture and consistency (Gurav et al., 2020).
       
One important factor influencing the stability and shelf life of ghee products is their moisture level. Being less in moisture content is generally preferable since it lowers the chance of lipid oxidation and microbial development (Nekera et al., 2023). An analysis of the effects of the herbal enrichment on moisture levels can be obtained by contrasting regular cow ghee with ghee supplemented with ashwagandha powder. Research on lipid stability and microbial development in dairy products can be used to examine how moisture content affects the quality of ghee (Pena-Serna and Restrepo-Betancur, 2020). The main component of ghee is fat and the amount of fat present is a significant indicator of its nutritional worth (Nekera et al., 2023).
       
Cow ghee has a characteristic golden-yellow color because of the presence of carotenoids (Misra et al., 2023). For the color attribute, it has been found that ghee prepared from Ashwagandha powder had a stronger tendency toward reddish brown. According to (Saha et al., 2012), ghee’s dark brown color may be caused by tannins contained in the aqueous extract of Ashwagandha root powder. Ashwagandha ghee’s lower color and appearance scores indicate a noticeable difference from control ghee. The incorporation of ashwagandha extracts may cause the ghee’s color to change, which consumers may notice. This result is corroborated by research on how herbal extracts affect food products’ visual characteristics (Gurav et al., 2020).
       
Both control ghee and Ashwagandha ghee have similar overall acceptability values, although the little differences in each metric. This implies that minor variations in flavor, texture and appearance do not considerably impact consumers’ overall acceptance of the products. The strong approval scores suggest that customers are still receptive to Ashwagandha ghee.
       
The number of carboxylic acid groups in fatty acid compounds is determined by the term “acid value.” Over some time, as oil or fat oxidizes, triglycerides are transformed into fatty acids and glycerol, increasing the total acid value. It can be explained by the presence of acidic phyto-constituents, the catalytic influence of iron (from the manufacturing vessel), or the production of free fatty acids from triglycerides present in ghee (Gurav et al., 2020). These may be responsible for the greater acidity of prepared ashwagandha ghee during storage when compared to control ghee (Gurav et al., 2020).
       
The storage condition affects the lipid oxidation of fat, oils, or fatty acids (Catalá, 2010). The level of lipid peroxidation, or auto-oxidation, in unsaturated fats and oils, is the primary indicator of rancidity and is measured in terms of peroxide value (Pena-Serna and Restrepo-Betancur, 2020). High acidity indicates a high peroxide value because free unsaturated acids oxidize more quickly and easily than identical acids in intact glycerides (Oluremi et al., 2013). It can be presumed that the maximum oxidative degradation of ghee is caused by the catalytic activity of iron and/or high temperature (Kochhar, 2016). It showed that the ashwagandha root provided a degree of protection against oxidative and catalytic damage as well as the production of peroxide (Gurav et al., 2020). In our study, we found similar results to a research paper by (Siddiq et al., 2005). They discovered that when they added extracts from the Moringa oleifera plant to sunflower oil, it significantly prevented the formation of harmful substances called peroxides when the oil was exposed to unfavorable conditions that speed up spoilage. Among the different extracts they tested, the one made with 80% methanol was the most effective for keeping the peroxide levels low (Siddiq et al., 2005). The reason for these variations in results when using different solvents to make extracts could be that Moringa oleifera contains both water-soluble and fat-soluble antioxidants and these compounds work differently. Interestingly, in some parts of southern India, people use fresh Moringa oleifera leaves when making ghee from cow and buffalo milk. They do this to help the ghee last longer on the shelf. So, our study aligns with Siddiq’s findings about the Moringa oleifera extracts being good at preserving oil quality and it also highlights a traditional practice in southern India where these leaves are used to extend the shelf life of ghee (Siddhuraju and Becker, 2003). The study by Merai et al., (2003), found that adding 0.6 percent of a special fraction of Tulsi leaves powder treated with silica gel charcoal to ghee was better at preserving the ghee’s quality than using a chemical antioxidant like BHA at a concentration of 0.02 per cent. It was more effective in preventing the ghee from getting rancid until it reached a peroxide level of 5 milliequivalents of peroxide oxygen. These studies favor the research that by adding the ashwagandha extract we could able to extend the shelf life of the herb-enriched ghee.
       
In a study examining how adding an extract from the herb Asparagus racemosus, also known as Shatavari, affects the storage stability of ghee, it was discovered that ghee samples containing the ethanolic extract of Shatavari demonstrated stronger antioxidant activity (Pawar et al., 2012). The ethanolic extract of Shatavari was more effective in preserving the quality of the ghee and preventing it from becoming rancid, both at the beginning and the end of the storage period, compared to the aqueous extract of the same herb. This suggests that using the ethanolic extract of Shatavari could be a better choice for enhancing the shelf life and stability of ghee. This means that these herbal extracts were better at neutralizing harmful DPPH radicals both when the ghee was fresh (on day zero) and even after it had undergone oxidation (at the end of 21 days) at a temperature of 80±10°C.

In this study, herbal ghee containing Ashwagandha may result in a functional food item with improved antioxidant qualities and a changed nutritional makeup. These results are significant for the dairy industry since ashwagandha’s great stability of sensory qualities and bioactive components allow it to be further incorporated into a variety of dairy-based products. Furthermore, the sensory characteristics of ghee showed that the incorporation of ashwagandha did not significantly alter the texture and body, color and appearance, flavor and mouthfeel, or overall acceptability. Ashwagandha ghee is a promising newcomer to the continually expanding field of functional foods, combining ancient knowledge with modern nutritional science.

There is no conflict of interest among authors.